A Human 3D Cardiomyocyte Risk Model to Study the Cardiotoxic Influence of X-rays and Other Noxae in Adults.

The heart tissue is a potential target of various noxae contributing to the onset of cardiovascular diseases. However, underlying pathophysiological mechanisms are largely unknown. Human stem cell-derived models are promising, but a major concern is cell immaturity when estimating risks for adults. In this study, 3D aggregates of human embryonic stem cell-derived cardiomyocytes were cultivated for 300 days and characterized regarding degree of maturity, structure, and cell composition. Furthermore, effects of ionizing radiation (X-rays, 0.1-2 Gy) on matured aggregates were investigated, representing one of the noxae that are challenging to assess. Video-based functional analyses were correlated to changes in the proteome after irradiation. Cardiomyocytes reached maximum maturity after 100 days in cultivation, judged by α-actinin lengths, and displayed typical multinucleation and branching. At this time, aggregates contained all major cardiac cell types, proven by the patch-clamp technique. Matured and X-ray-irradiated aggregates revealed a subtle increase in beat rates and a more arrhythmic sequence of cellular depolarisation and repolarisation compared to non-irradiated sham controls. The proteome analysis provides first insights into signaling mechanisms contributing to cardiotoxicity. Here, we propose an in vitro model suitable to screen various noxae to target adult cardiotoxicity by preserving all the benefits of a 3D tissue culture.

Eficácia de um produto à base de oxigênio ativo no tratamento não cirúrgico da Mucosite peri-implantar / Efficacy of an active oxygen-based product in the non-surgical treatment of peri-implant mucositis

A mucosite peri-implantar é uma condição inflamatória sem perda de tecido ósseo, que ocorre nos tecidos moles ao redor do implante. O tratamento não-cirúrgico combina a terapia básica associada ou não ao uso de agentes químicos. O objetivo desse estudo foi avaliar a eficácia do oxigênio ativo (Bluem®) como coadjuvante químico no tratamento não-cirúrgico da mucosite peri­implantar em um protocolo de tratamento não­cirúrgico. Sendo assim, 20 implantes foram tratados, 9 no grupo teste (Bluem®) e 11 no grupo controle (placebo), em indivíduos reabilitados com implantes. Desta forma, os pacientes foram divididos aleatoriamente em grupo teste e grupo controle. Os parâmetros clínicos peri-implantares de índice de placa visível (IPV), índice de sangramento gengival (ISG), profundidade de sondagem (PS), sangramento à sondagem (SS) e mucosa ceratinizada foram avaliados no baseline e em diferentes períodos após o tratamento. Para análise estatística, foram utilizados os testes de Mann-Whitney, Friedman, Wilcoxon e Exato de Fisher com nível de significância de 5%. Os resultados mostraram que houve diminuição significativamente estatística para: IPV geral (56,81% para 27,27%) e ISG geral (32,95% para 28,41%) do grupo teste; IPV geral (42,86% para 15,18%) e ISG geral (31,25% para 2,32%) do grupo controle; IPV do implante no grupo controle (21,42% para 0%); profundidade de sondagem para o grupo teste (2,83mm para 2mm) e SS para o grupo controle (33,33% para 16,67%). No entanto, não houve diferenças significativas entre os dois grupos quando foram comparados em cada período. Pode-se concluir que o Bluem pode ser utilizado como adjuvante químico no tratamento da mucosite peri-implantar, entretanto a sua real eficácia necessita de estudos adicionais para comprovação científica (AU).

Peri-implant mucositis is an inflammatory condition without loss of bone tissue, which occurs in the soft tissues around the implant. Non-surgical treatment combines basic periodontal therapy with or without the use of chemical agents. The objective of this study was to evaluate the efficacy of the active oxygen (Bluem®) as a chemical adjuvant in the non-surgical treatment of peri-implant mucositis. Twenty implants in 9 patients were ramdomly treated, 9 in the test group (Bluem®) and 11 in the control group (placebo), in individuals rehabilitated with dental implants. Peri-implant clinical parameters of visible plaque index (VPI), gingival bleeding index (GBI), probing depth (PD), bleeding on probing (BOP) and keratinized mucosa were evaluated at baseline and one and six months. For the statistical analysis, Mann-Whitney, Friedman, Wilcoxon and Fisher's Exact tests were used with a significance level of 5%. The results showed that there was a statistical decrease for full mouth VPI (56.81% to 27.27%) and full mouth GBI (32.95% to 28.41%) of the test group; full mouth VPI (42.86% to 15.18%) and full mouth GBI (31.25% to 2.32%) of the control group; Implant VPI in the control group (21.42% to 0%); probing depth for the test group (2.83mm to 2mm) and BOP for the control group (33.33% to 16.67%). However, there were no significant differences between the two groups. It can be concluded that Bluem can be used as a chemical adjuvant in the treatment of peri-implant mucositis (AU).

Reduction in activity by noxious chemical stimulation is ameliorated by immersion in analgesic drugs in zebrafish.

Research has recently demonstrated that larval zebrafish show similar molecular responses to nociception to those of adults. Our study explored whether unprotected larval zebrafish exhibited altered behaviour after exposure to noxious chemicals and screened a range of analgesic drugs to determine their efficacy to reduce these responses. This approach aimed to validate larval zebrafish as a reliable replacement for adults as well as providing a high-throughput means of analysing behavioural responses. Zebrafish at 5 days post-fertilization were exposed to known noxious stimuli: acetic acid (0.01%, 0.1% and 0.25%) and citric acid (0.1%, 1% and 5%). The behavioural response of each was recorded and analysed using novel tracking software that measures time spent active in 25 larvae at one time. Subsequently, the efficacy of aspirin, lidocaine, morphine and flunixin as analgesics after exposure to 0.1% acetic acid was tested. Larvae exposed to 0.1% and 0.25% acetic acid spent less time active, whereas those exposed to 0.01% acetic acid and 0.1-5% citric acid showed an increase in swimming activity. Administration of 2.5 mg l-1 aspirin, 5 mg l-1 lidocaine and 48 mg l-1 morphine prevented the behavioural changes induced by acetic acid. These results suggest that larvae respond to a noxious challenge in a similar way to adult zebrafish and other vertebrates and that the effect of nociception on activity can be ameliorated by using analgesics. Therefore, adopting larval zebrafish could represent a direct replacement of a protected adult fish with a non-protected form in pain- and nociception-related research.

Intestinal injury in the rat model of 17α-ethynylestradiol-induced intrahepatic cholestasis.

OBJECTIVE: Although the intimate relationship between liver and gut has been previously reported under physiological and pathological conditions, intestinal involvement in the process of intrahepatic cholestasis of pregnancy remains unclear. The aim of this study was to investigate intestinal changes in 17α-ethynylestradiol (EE)-induced cholestatic rat model. METHODS: Liver injury was assessed by HE stain and serum biochemical parameters were measured. Intestinal transit was determined using ink marks. Neuronal protein expressions in the intestine were analyzed by Western blot. RESULTS: EE treatment induced liver damage, including severe bile duct hyperplasia, portal edema, portal infiltration, a loss of hepatic structure in periportal areas and increased serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and total bilirubin. Large areas of inflammatory cell infiltration and increased myeloperoxidase activity were observed in the intestine of EE-induced cholestatic rats. The EE-treated group showed increased intestinal transit and malondialdehyde levels, while the glutathione content and superoxide dismutase activity were notably decreased, together with decreased protein gene product 9.5 and neuronal nitric oxide synthase expression in the ileum and colon. Furthermore, choline acetyltransferase expression was significantly decreased in the ileum, whereas no change was observed in the colon of EE-treated rats. CONCLUSION: EE-induced liver damage is associated with oxidative stress, inflammation and neural loss in the intestine, which may lead to altered intestinal motility.

Enriquecimiento-mejora en la eficacia de la realización de la reacción en cadena de la polimerasa de patógenos en alimentos / Enrichment-improvement in the effectiveness of the implementation of the polymerase chain reaction of pathogens in food

Objetivo. Mejorar la eficacia de los ensayos cualitativos por reacción en cadena de la polimerasa (PCR) de patógenos en alimentos. Al realizar un enriquecimiento consistente en hacer una dilución inferior a la 1/10 se pretende adelantar en tiempo, reducir el gasto de medios, se procura posibilitar el completar hasta el 100% las muestras a realizar en una PCR y no disminuir la carga celular diana del alimento. Métodos. Investigación de la presencia o ausencia de una bacteria patógena en un alimento y su concentración por mililitro de disolución, siguiendo el método tradicional a distintas diluciones, en dos matrices. Resultados. Los resultados obtenidos en el experimento con un patógeno en alimentos, evidencia que con diluciones inferiores a 1/10, la concentración bacteriana para un mínimo de horas de incubación adelanta la detección por PCR del ácido desoxirribonucleico (ADN) diana, con valores de ufc/ml por encima de 1x103 . Por tanto el tiempo de incubación en un caldo de enriquecimiento se acorta, así como los volúmenes de las disoluciones a incubar. Y de este modo solo es necesario un operario y una estufa de incubación para realizar todas las tareas. Conclusiones. Mayor eficacia al necesitarse menos medios materiales y horas de trabajo. Realización de mayor número de muestras en una misma jornada de trabajo (se posibilita realizar el total de muestras que admite el termociclador por cada puesta en funcionamiento), se acorta el tiempo de análisis en siete horas como mínimo

Objective. Improve the effectiveness of the tests by qualitative polymerase chain reaction (PCR) of pathogens in food. When performing a enrichment consists in making a lower dilution to the 1/10 is intends to bring forward in time, reduce the cost of media, it seeks to enable the complete up to 100% of the samples to perform in a PCR and does not decrease the load cell Diana of the food. Methods. Investigation of the presence or absence of a pathogenic bacterium in food and its concentration per millilitre of dissolution, following the traditional method in different dilutions, in two different types of food. Results. The results obtained in the experiment with a pathogen in food, evidence that using dilutions lower than 1/10, the bacterial concentration for a minimum number of hours of incubation anticipates the detection by PCR of deoxyribonucleic acid (DNA) target, with values of cfu/ml above 1x103 . Therefore the incubation time in an enrichment broth and the volumes of the solutions are reduced. And it is only needed one operator and one incubator for all tasks. Conclusions. The efficacy increases because are needed less material and working hours. Testing more samples in the same working day (makes possible to perform all samples that supports the thermal cycler for each start-up) the analysis time decreases in seven hours at least

Avaliação de extratos de quebra- pedra (Phyllanthus sp) frente à patógenos causadores de infecções no trato urinário / Evaluation of Phyllanthus sp extracts in face of Pathogen Causers of Urinary Treat Infections

RESUMO O trato urinário normalmente é estéril, no entanto, pode ser contaminado por agentes etiológicos provenientes da microbiota intestinal, dentre os mais comuns pode-se destacar a Escherichia coli. Os microrganismos estão se tornando cada vez mais resistentes a múltiplos antimicrobianos. É notória, portanto, a necessidade de encontrar novas substâncias com propriedades antimicrobianas. Portanto, foram avaliados diferentes extratos de Phyllanthus sp, frente a microrganismos causadores de infecções no trato urinário e comparadas as técnicas de hole-plate e disco difusão. Para ambas as técnicas avaliadas, o extrato de 72 horas mostrou melhor atividade antimicrobiana, na técnica de disco difusão, a bactéria mais sensível foi o Staphylococcus saprophyticcus, que apresentou CMI (Concentração Mínima Inibitória) de 15,84 mg/mL. Com a utilização da técnica de hole-plate, a bactéria mais sensível observada foi Staphylococcus aureus, com valor de CMI igual ao reportado anteriormente. Foi observado que os extratos alcoólicos obtiveram maior eficiência em relação às infusões, que a técnica de hole-plate revelou ser mais eficiente que a técnica de disco difusão e que os cocos Gram positivos foram mais susceptíveis quando comparadas aos bacilos Gram negativos e fungos.

ABSTRACT Commonly, the urinary treat is sterile, but it can also be contaminated by etiological agents from the intestinal treat, of which the Escherichiacoli is the most common one. These microorganisms are increasingly becoming more resistant to multiple antibiotics. It became necessary to find new substances with antimicrobial properties, so the purpose of this paper is to evaluate different Phyllanthus sp extracts- in face of microorganisms which cause the urinary treat infections- and compare it to the hole-plate and disk diffusion techniques. The 72 hours extraction showed better antimicrobial activity in both methods. Using disk diffusion, the most sensitive bacterium was the Staphylococcus saprophyticcus, with the MIC of 15,84 mg/mL. While using the technique of hole-plate, the most sensitive bacterium was the Staphylococcus aureus, with the same MIC of the previous cited bacterium. This study showed that the alcoholic extracts were more efficient than the infusions. It can also be observed that the hole-plate technique seems to be more efficient than the disk diffusion one, and the Gram positive cocci bacteria were more sensitive than the Gram negative bacilli and fungi.

Analysis of the role of von Willebrand factor, platelet glycoprotein VI-, and α2ß1-mediated collagen binding in thrombus formation.

Rare missense mutations in the von Willebrand factor (VWF) A3 domain that disrupt collagen binding have been found in patients with a mild bleeding phenotype. However, the analysis of these aberrant VWF-collagen interactions has been limited. Here, we have developed mouse models of collagen-binding mutants and analyzed the function of the A3 domain using comprehensive in vitro and in vivo approaches. Five loss-of-function (p.S1731T, p.W1745C, p.S1783A, p.H1786D, A3 deletion) and 1 gain-of-function (p.L1757A) variants were generated in the mouse VWF complementary DNA. The results of these various assays were consistent, although the magnitude of the effects were different: the gain-of-function (p.L1757A) variant showed consistent enhanced collagen binding whereas the loss-of-function mutants showed variable degrees of functional deficit. We further analyzed the impact of direct platelet-collagen binding by blocking glycoprotein VI (GPVI) and integrin α2ß1 in our ferric chloride murine thrombosis model. The inhibition of GPVI demonstrated a comparable functional defect in thrombosis formation to the VWF(-/-) mice whereas α2ß1 inhibition demonstrated a milder bleeding phenotype. Furthermore, a delayed and markedly reduced thrombogenic response was still evident in VWF(-/-), GPVI, and α2ß1 blocked animals, suggesting that alternative primary hemostatic mechanisms can partially rescue the bleeding phenotype associated with these defects.

Modality-specific thalamocortical inputs instruct the identity of postsynaptic L4 neurons.

During development, thalamocortical (TC) input has a critical role in the spatial delineation and patterning of cortical areas, yet the underlying cellular and molecular mechanisms that drive cortical neuron differentiation are poorly understood. In the primary (S1) and secondary (S2) somatosensory cortex, layer 4 (L4) neurons receive mutually exclusive input originating from two thalamic nuclei: the ventrobasalis (VB), which conveys tactile input, and the posterior nucleus (Po), which conveys modulatory and nociceptive input. Recently, we have shown that L4 neuron identity is not fully committed postnatally, implying a capacity for TC input to influence differentiation during cortical circuit assembly. Here we investigate whether the cell-type-specific molecular and functional identity of L4 neurons is instructed by the origin of their TC input. Genetic ablation of the VB at birth resulted in an anatomical and functional rewiring of Po projections onto L4 neurons in S1. This induced acquisition of Po input led to a respecification of postsynaptic L4 neurons, which developed functional molecular features of Po-target neurons while repressing VB-target traits. Respecified L4 neurons were able to respond both to touch and to noxious stimuli, in sharp contrast to the normal segregation of these sensory modalities in distinct cortical circuits. These findings reveal a behaviourally relevant TC-input-type-specific control over the molecular and functional differentiation of postsynaptic L4 neurons and cognate intracortical circuits, which instructs the development of modality-specific neuronal and circuit properties during corticogenesis.

Accelerated premature stress-induced senescence of young annulus fibrosus cells of rats by high glucose-induced oxidative stress.

PURPOSES: Diabetes mellitus (DM) is thought to be an important aetiological factor in intervertebral disc degeneration. A glucose-mediated increase of oxidative stress is a major causative factor in development of diseases associated with DM. The aim of this study was to investigate the effect of high glucose on mitochondrial damage, oxidative stress and senescence of young annulus fibrosus (AF) cells. METHODS: AF cells were isolated from four-week-old young rats, cultured, and placed in either 10 % FBS (normal control) or 10 % FBS plus two different high glucose concentrations (0.1 M and 0.2 M) (experimental conditions) for one and three days. We identified and quantified the mitochondrial damage and reactive oxygen species (ROS) (oxidative stress). We also identified and quantified the occurrence of senescence and telomerase activity. Finally, the expressions of proteins were determined related to replicative senescence (p53-p21-pRB) and stress-induced senescence (p16-pRB). RESULTS: Two high glucoses enhanced the mitochondrial damage in young rat AF cells, which resulted in an excessive generation of ROS in a dose- and time-dependent manner for one and three days compared to normal control. Two high glucose concentrations increased the occurrence of senescence of young AF cells in a dose- and time-dependent manner. Telomerase activity declined in a dose- and time-dependent manner. Both high glucose treatments increased the expressions of p16 and pRB proteins in young rat AF cells for one and three days. However, compared to normal control, the expressions of p53 and p21 proteins were decreased in young rat AF cells treated with both high glucoses for one and three days. CONCLUSIONS: The present study demonstrated that high glucose-induced oxidative stress accelerates premature stress-induced senescence in young rat AF cells in a dose- and time-dependent manner rather than replicative senescence. These results suggest that prevention of excessive generation of oxidative stress by strict blood glucose control could be important to prevent or to delay premature intervertebral disc degeneration in young patients with DM.

Regulation of HFE expression by poly(ADP-ribose) polymerase-1 (PARP1) through an inverted repeat DNA sequence in the distal promoter.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron overload among Caucasians of northern European descent. Over 85% of all cases with HH are due to mutations in the hemochromatosis protein (HFE) involved in iron metabolism. Although the importance in iron homeostasis is well recognized, the mechanism of sensing and regulating iron absorption by HFE, especially in the absence of iron response element in its gene, is not fully understood. In this report, we have identified an inverted repeat sequence (ATGGTcttACCTA) within 1700bp (-1675/+35) of the HFE promoter capable to form cruciform structure that binds PARP1 and strongly represses HFE promoter. Knockdown of PARP1 increases HFE mRNA and protein. Similarly, hemin or FeCl3 treatments resulted in increase in HFE expression by reducing nuclear PARP1 pool via its apoptosis induced cleavage, leading to upregulation of the iron regulatory hormone hepcidin mRNA. Thus, PARP1 binding to the inverted repeat sequence on the HFE promoter may serve as a novel iron sensing mechanism as increased iron level can trigger PARP1 cleavage and relief of HFE transcriptional repression.

Desmolaris, a novel factor XIa anticoagulant from the salivary gland of the vampire bat (Desmodus rotundus) inhibits inflammation and thrombosis in vivo.

The identity of vampire bat saliva anticoagulant remained elusive for almost a century. Sequencing the salivary gland genes from the vampire bat Desmodus rotundus identified Desmolaris as a novel 21.5-kDa naturally deleted (Kunitz 1-domainless) form of tissue factor pathway inhibitor. Recombinant Desmolaris was expressed in HEK293 cells and characterized as a slow, tight, and noncompetitive inhibitor of factor (F) XIa by a mechanism modulated by heparin. Desmolaris also inhibits FXa with lower affinity, independently of protein S. In addition, Desmolaris binds kallikrein and reduces bradykinin generation in plasma activated with kaolin. Truncated and mutated forms of Desmolaris determined that Arg32 in the Kunitz-1 domain is critical for protease inhibition. Moreover, Kunitz-2 and the carboxyl-terminus domains mediate interaction of Desmolaris with heparin and are required for optimal inhibition of FXIa and FXa. Notably, Desmolaris (100 µg/kg) inhibited FeCl3-induced carotid artery thrombus without impairing hemostasis. These results imply that FXIa is the primary in vivo target for Desmolaris at antithrombotic concentrations. Desmolaris also reduces the polyphosphate-induced increase in vascular permeability and collagen- and epinephrine-mediated thromboembolism in mice. Desmolaris emerges as a novel anticoagulant targeting FXIa under conditions in which the coagulation activation, particularly the contact pathway, plays a major pathological role.

Elevated prothrombin promotes venous, but not arterial, thrombosis in mice.

OBJECTIVE: Individuals with elevated prothrombin, including those with the prothrombin G20210A mutation, have increased risk of venous thrombosis. Although these individuals do not have increased circulating prothrombotic biomarkers, their plasma demonstrates increased tissue factor-dependent thrombin generation in vitro. The objectives of this study were to determine the pathological role of elevated prothrombin in venous and arterial thrombosis in vivo, and distinguish thrombogenic mechanisms in these vessels. APPROACH AND RESULTS: Prothrombin was infused into mice to raise circulating levels. Venous thrombosis was induced by electrolytic stimulus to the femoral vein or inferior vena cava ligation. Arterial thrombosis was induced by electrolytic stimulus or ferric chloride application to the carotid artery. Mice infused with prothrombin demonstrated increased tissue factor-triggered thrombin generation measured ex vivo, but did not have increased circulating prothrombotic biomarkers in the absence of vessel injury. After venous injury, elevated prothrombin increased thrombin generation and the fibrin accumulation rate and total amount of fibrin ≈ 3-fold, producing extended thrombi with increased mass. However, elevated prothrombin did not accelerate platelet accumulation, increase the fibrin accumulation rate, or shorten the vessel occlusion time after arterial injury. CONCLUSIONS: These findings reconcile previously discordant findings on thrombin generation in hyperprothrombinemic individuals measured ex vivo and in vitro, and show elevated prothrombin promotes venous, but not arterial, thrombosis in vivo.

A platelet target for venous thrombosis? P2Y1 deletion or antagonism protects mice from vena cava thrombosis.

A role for platelets in the pathogenesis of venous thrombosis was suggested by clinical and preclinical studies. However, examination of the platelet receptor, P2Y1, in this area has been limited. The goal of the current study was to examine effects of P2Y1 deletion, or selective antagonism with MRS2500, in oxidative venous thrombosis in mice. The P2Y12 antagonist, clopidogrel, was included as a reference agent. Anesthetized C57BL/6 or genetically modified mice underwent 3.5 or 5 % FeCl(3)-induced vena cava thrombosis. Pharmacokinetic properties of MRS2500 were defined for dose selection. Platelet aggregation and renal or tail bleeding times (BT) were measured to put antithrombotic effects into perspective. P2Y1 deletion significantly reduced (p < 0.001) venous thrombus weight by 74 % in 3.5 % FeCl(3) injury compared to P2Y1(+/+) littermates. MRS2500 (2 mg/kg, i.v.) significantly decreased (p < 0.001) thrombus weight 64 % in C57BL/6 mice. In the more severe 5 % FeCl(3)-induced injury model, thrombus weight significantly (p < 0.001) decreased 68 % in P2Y1(-/-) mice versus P2Y1(+/+) mice, and MRS2500 (2 mg/kg) was also beneficial (54 % decrease, p < 0.01). Renal BT doubled in P2Y1(-/-) versus P2Y1(+/+) mice, and increased threefold with MRS2500 compared to vehicle. Tail BT was markedly prolonged in P2Y1(-/-) mice (7.9X) and in C57BL/6 mice given MRS2500. The current study demonstrates that P2Y1 deletion or antagonism significantly reduced venous thrombosis in mice, suggesting that P2Y1 receptors play a role in the pathogenesis of venous thrombosis, at least in this species. However as with many antithrombotic agents the benefit comes at the potential price of an increase in provoked bleeding times.

Natural organic compounds as alternative to methyl bromide for nematodes control.

Thirty-three organic acids and furfural metabolites were examined for their nematicidal activity against plant-parasitic, free-living and predacious nematodes. Propionic acid, 2-methylhexanoic acid, lactic acid, maleic acid, and furic acid were the most effective nematicides among normal chain organic acids, branched organic acids, hydroxy/keto-acids, dicarboxylic acids and furfural metabolites, respectively. Seven of the tested compounds were found to have more than 90% mortality thus designating them as highly active nematicides. Of the highly active tested compounds, an average octanol/water log P of 0.97 was observed with a range from 0.28 to 2.64, and a Henry's Law constant averaging 2.6 x 10(- 7) atm.m3/mole. Tested chemicals with minor or low nematicidal activity showed an average log P of 1.76 with a range from 0.15 to 3.42 and a Henry's Law constant averaging 16.6 x 10(- 7) atm.m3/mole.

Serglycin proteoglycan deletion induces defects in platelet aggregation and thrombus formation in mice.

Serglycin (SG), the hematopoietic cell secretory granule proteoglycan, is crucial for storage of specific secretory proteins in mast cells, neutrophils, and cytotoxic T lymphocytes. We addressed the role of SG in platelets using SG-/- mice. Wild-type (WT) but not SG-/- platelets contained chondroitin sulfate proteoglycans. Electron microscopy revealed normal alpha-granule structure in SG-/- platelets. However, SG-/- platelets and megakaryocytes contained unusual scroll-like membranous inclusions, and SG-/- megakaryocytes showed extensive emperipolesis of neutrophils. SG-/- platelets had reduced ability to aggregate in response to low concentrations of collagen or PAR4 thrombin receptor agonist AYPGKF, and reduced fibrinogen binding after AYPGKF, but aggregated normally to ADP. 3H-serotonin and ATP secretion were greatly reduced in SG-/- platelets. The alpha-granule proteins platelet factor 4, beta-thromboglobulin, and platelet-derived growth factor were profoundly reduced in SG-/- platelets. Exposure of P-selectin and alphaIIb after thrombin treatment was similar in WT and SG-/- platelets. SG-/- mice exhibited reduced carotid artery thrombus formation after exposure to FeCl3. This study demonstrates that SG is crucial for platelet function and thrombus formation. We propose that SG-/- platelet function deficiencies are related to inadequate packaging and secretion of selected alpha-granule proteins and reduced secretion of dense granule contents critical for platelet activation.

In vivo EPR estimation of bilateral hippocampal antioxidant ability of rats with epileptogenesis induced by amygdalar FeCl3 microinjection.

PURPOSE: To measure the neural antioxidant function in the hippocampus of rats with epileptogenesis induced by microinjection of FeCl3 into the amygdala using the decay rate of the nitroxide radical as estimated by L-band electron paramagnetic resonance (EPR) spectroscopy. MATERIALS AND METHODS: Region-selected intensity determination (RSID) was used for the estimation of the nitroxide decay ratio. It is possible to estimate the in vivo hippocampal antioxidant ability using the half-life of the EPR signal of the blood-brain barrier-permeable nitroxide radical. Rats were microinjected with aqueous FeCl3 into the right amygdaloid body. Recording from chronically implanted depth electrodes showed the development of spike discharges with recurrent seizures arising from amygdalar regions with propagation into both hippocampi. Rats with unilateral aqueous FeCl3 lesions were injected systemically with the nitroxide radical and then had EPR for RSID estimation at 5, 15, and 30 days after the iron salt injection. RESULTS: The in vivo antioxidant ability of the dorsal hippocampus was significantly decreased bilaterally in animals with FeCl3-induced seizures when compared to the control. CONCLUSION: Neural antioxidant function in the hippocampi of rats with chronic seizures induced by amygdalar FeCl3 was decreased early and both ipsilaterally and bilaterally.

The 3D structure of protein phosphatase 2A: new insights into a ubiquitous regulator of cell signaling.

Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase implicated in cancer. Three new crystal structures of PP2A show how it interacts with inhibitory toxins and with one of its regulatory subunits. The structures also explain how specific site mutations may lead to cancer and suggest a novel role for PP2A methylation in the formation of PP2A holoenzymes.

Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines.

The nervous system senses peripheral damage through nociceptive neurons that transmit a pain signal. TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels and is expressed in nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants. How such diverse stimuli activate TRPA1 is not known. We observed that most compounds known to activate TRPA1 are able to covalently bind cysteine residues. Here we use click chemistry to show that derivatives of two such compounds, mustard oil and cinnamaldehyde, covalently bind mouse TRPA1. Structurally unrelated cysteine-modifying agents such as iodoacetamide (IA) and (2-aminoethyl)methanethiosulphonate (MTSEA) also bind and activate TRPA1. We identified by mass spectrometry fourteen cytosolic TRPA1 cysteines labelled by IA, three of which are required for normal channel function. In excised patches, reactive compounds activated TRPA1 currents that were maintained at least 10 min after washout of the compound in calcium-free solutions. Finally, activation of TRPA1 by disulphide-bond-forming MTSEA is blocked by the reducing agent dithiothreitol (DTT). Collectively, our data indicate that covalent modification of reactive cysteines within TRPA1 can cause channel activation, rapidly signalling potential tissue damage through the pain pathway.

Emissions and distribution of methyl bromide in field beds applied at two rates and covered with two types of plastic mulches.

A field experiment was conducted to compare two plastic mulches and two application rates on surface emissions and subsurface distribution of methyl bromide (MBr) in field beds in Florida. Within 30 minutes after injection of MBr to 30 cm depth, MBr had diffused upward to soil surface in all beds covered with polyethylene film (PE) or virtually impermeable film (VIF) and applied at a high rate (392 kg/ha) and a low rate (196 kg/ha). Due to the highly permeable nature of PE, within 30 minutes after injection, MBr volatilized from the bed surfaces of the two PE-covered beds into the atmosphere. The amount of volatilization was greater for the high rate-treatment bed. On the other hand, volatilization of MBr from the bed surfaces of the two VIF-covered beds were negligible. Volatilization losses occurred from the edges of all the beds covered with PE or VIF and were greater from the high rate-treatment beds. Initial vertical diffusion of MBr in the subsurface of the beds covered with PE or VIF was mainly upward, as large concentrations of MBr were detected from near bed surfaces to 20 cm depth in these beds 30 minutes after injection and little or no MBr was found at 40 cm depth. The two VIF-covered beds exhibited greater MBr concentrations and longer resident times in the root zone (0.5-40 cm depth) than corresponding PE-covered beds. Concentrations of MBr in the root zone of the high rate-treatment beds were 3.6-6.1 times larger than the low rate-treatment beds during the first days after application. In conclusion, VIF promoted retention of MBr in the root zone and, if volatilization loss from bed edges can be blocked, volatilization loss from VIF-covered beds should be negligible.

Calcium-permeable acid-sensing ion channel is a molecular target of the neurotoxic metal ion lead.

Acid-sensing ion channels (ASICs) are emerging as fundamental players in the regulation of neural plasticity and in pathological conditions. Here we showed that lead (Pb2+), a well known neurotoxic metal ion, reversibly and concentration-dependently inhibited ASIC currents in the acutely dissociated spinal dorsal horn and hippocampal CA1 neurons of rats. In vitro expression of ASIC subunits in combination demonstrated that both ASIC1 and -3 subunits were sensitive to Pb2+. Mechanistically, Pb2+ reduced the pH sensitivity of ASICs independent of membrane voltage change. Moreover, Pb2+ inhibited the ASIC-mediated membrane depolarization and the elevation of intracellular Ca2+ concentration. In addition, we compared the effect of Pb2+ with that of Ca2+ or amiloride to explore the possible interactions of Pb2+ and Ca2+ in regulating ASICs, and we found that Pb2+ inhibited ASIC currents independent of the amiloride/Ca2+ blockade. Because ASIC1b and -3 subunits are mainly expressed in peripheral neurons, our data identified ASIC1a-containing Ca2+-permeable ASIC as a novel central target of Pb2+ action, which may contribute to Pb2+ neurotoxicity.